Application of OsMYB71 gene in improvement of saline-alkaline tolerance of rice

Abstract

The invention discloses application of an OsMYB71 gene in improvement of saline-alkaline tolerance of rice, and belongs to the technical field of plant stress resistance. The invention aims to improve the saline-alkaline stress tolerance of rice. The invention provides an application of OsMYB71 amino acid in regulation and control of salt resistance and / or alkali resistance of rice. The sequence of the OsMYB71 amino acid is shown as SEQ ID NO.8. The invention also provides an application of the OsMYB71 amino acid in regulation and control of salt resistance and / or alkali resistance of rice. And a theoretical basis is provided for saline-alkaline stress tolerance of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant stress resistance technology, specifically involving the application of the OsMYB71 gene in improving the salt and alkali tolerance of rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, but its yield reduction due to environmental factors is becoming increasingly serious. Soil salinization is one of the major abiotic stress factors hindering normal plant growth, causing not only ecological degradation but also severely restricting agricultural production. Currently, the global area affected by salinization exceeds 950 million hectares, with an annual growth rate of over 1 million hectares. In China, the area of ​​saline-alkali soil is approximately 100 million hectares (about 1.5 billion mu), concentrated in Northeast China, North China, Northwest China, and parts of the coastal region. The Songnen Plain is one of the areas with concentrated and contiguous saline-alkali land in my country, covering approximately 7.65 million hectares, with soil types classified as soda saline-alkali soil (mainly including NaHCO3 and Na2CO3), accounting for 7.7% of the total saline-alkali land area in the country. Research shows that rice cultivation on saline-alkali land is an effective measure for improving and utilizing saline-alkali land. Furthermore, approximately 6.67 million hectares of saline-alkali land in China have the potential for rice cultivation, representing significant application potential. Therefore, improving the salt and alkali tolerance of rice varieties and effectively managing more saline-alkali land is of great significance to ensuring my country's food security.

[0003] Alkali tolerance determines whether rice can maintain normal tillering, heading, and grain filling under alkaline stress. Rice maintains ion homeostasis through a "rejection-chelation-efflux" strategy, ensuring that the rhizosphere Na+... + Loading into the xylem is hindered, and Na+ is transported by ion transport proteins. + Retention in roots and sheaths reduces Na in the aboveground parts + / K + Compared to other methods, alkaline stress helps maintain cell turgor pressure and photosynthetic rate; it activates osmotic regulatory genes to maintain osmotic balance, accumulates proline and soluble sugars, and stabilizes protein and membrane structures; it activates reactive oxygen species scavenging genes to deal with excess reactive oxygen species produced in cells after stress, maintaining the environmental conditions for normal cell function; and it transmits stress signals through hormones such as ABA, GA, and JA, participating in the operation of various regulatory systems. In general, alkaline signals are transmitted through a cascade of kinases, inducing transcription factor expression, regulating the antioxidant enzyme system and the synergistic network of ion transport proteins, improving plant survival rate under alkaline stress, providing nutritional support for tillering, fruiting, and grain filling under saline-alkali stress, and achieving stable yields in saline-alkali land. Summary of the Invention

[0004] The purpose of this invention is to improve the salt and alkali tolerance of rice.

[0005] This invention provides the application of OsMYB71 amino acid in regulating the salt and / or alkali tolerance of rice, wherein the OsMYB71 amino acid sequence is shown in SEQ ID NO.9.

[0006] This invention provides an application of the OsMYB71 gene in regulating the salt and / or alkali tolerance of rice, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0007] This invention provides the application of a recombinant vector containing the OsMYB71 gene in regulating the rice's tolerance to salt and / or alkali, wherein the nucleotide sequence of the OsMYB71 gene is shown in SEQ ID NO.8.

[0008] Further specifying, the starting vector is pEGOEPubi-H-GFP.

[0009] This invention provides the application of recombinant microbial cells containing the OsMYB71 gene in regulating the salt and / or alkali tolerance of rice, wherein the nucleotide sequence of the OsMYB71 gene is shown in SEQ ID NO.8.

[0010] To further specify, the originating microbial cell is either a prokaryotic or eukaryotic microbial cell.

[0011] Further, the conditions for alkali stress are 80 mM sodium bicarbonate; the conditions for salt stress are 120 mM sodium chloride.

[0012] This invention provides a breeding method for improving the salt and alkali tolerance of rice, the steps of which are as follows: Step 1: Amplify the OsMYB71 gene and then ligate it into the pCAMBIA1300-35S-EGFP vector to obtain the recombinant vector; the nucleotide sequence of the OsMYB71 gene is shown in SEQ ID NO.8; Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Transfect recombinant Agrobacterium into rice callus and culture to obtain transgenic rice.

[0013] To further specify, the primers used to amplify the OsMYB71 gene in step 1 are SEQ ID NO.6 and SEQ ID NO.7.

[0014] This invention provides a method for improving the salt and alkali tolerance of rice by overexpressing the OsMYB71 gene in rice and culturing it under conditions of 80mM sodium bicarbonate or 120mM sodium chloride.

[0015] Beneficial effects: OsMYB71 improves the alkali resistance of rice by promoting the accumulation of proline and clearing malondialdehyde content. Rice lines with OsMYB71 gene knockout at the bud and seedling stages showed significantly lower plant height, root length, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, chlorophyll content, and survival rate than wild type under salt-alkali stress. Attached Figure Description

[0016] Figure 1 Electrophoresis image of the amplified OsMYB71 gene; Figure 2 Image of the yl-hu-colored rice-LOC_Os07g37210-cas9 vector; Figure 3 The image shows the results of AscI restriction enzyme digestion detection of the yl-hu-CD-OsMYB71-cas9 vector. Figure 4 Electrophoresis image of transgenic T0 generation mutant plant; Figure 5 This is a sequence of sequencing results for a homozygous OsMYB71 mutant plant. Figure 6 Map of the OsMYB71 overexpression vector; Figure 7 Electrophoresis diagram for overexpression vector restriction enzyme digestion identification, M: DNA Marker (D5000), K: overexpression vector restriction enzyme digestion; Figure 8 Electrophoresis diagram of T0 generation transgenic plants for transgenic detection of overexpressing plants; M: DNA Marker (D2000); 1-12: T0 generation plants; N: negative control; P: positive control; Figure 9 This is the result of the qPCR test; Figure 10 Figure showing the results of alkali tolerance verification during the bud stage of transgenic OsMYB71; Figure 11 This is a graph showing the results of coleoptile length measurement during the budding stage; Figure 12 This is a graph showing the results of radicle length measurement during the budding stage; Figure 13 Figure showing the results of alkali tolerance verification of transgenic OsMYB71 seedlings; Figure 14 The graph shows the results of MDA content determination. Figure 15 The graph shows the results of proline content determination. Figure 16 This is a graph showing the results of peroxidase activity assay. Figure 17 The graph shows the results of H2O2 content determination. Figure 18 It is a graph showing the determination results of catalase enzyme activity; Figure 19 It is a graph showing the determination results of superoxide dismutase enzyme activity; Figure 20 It is a graph showing the DAB and NBT staining results; Figure 21 It is a graph showing the verification results of the salt tolerance function of transgenic OsMYB71 at the seedling stage. Specific implementation manners

[0017] Example 1. Construction of the CRISPR-CAS9 expression vector of the OsMYB71 gene The gRNA expression cassette with 1 target site was ligated to the backbone of the pYLCRISPR / Cas9-MT vector, and the constructed vector was yl-hu-color rice-LOC_Os07g37210 -cas9 ( Figure 2 ) vector. The types of the gene editing vectors are pYLsgRNA-OsU3 and pYLCRISPR / Cas9-MT vectors, which were both kindly provided by Professor Liu Yaoguang of South China Agricultural University and have been published: Ma X L, Zhang Q Y, Zhu Q L, et al. A Robust CRISPR / Cas9 System forConvenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants[J]. Molecular Plant, 2015, 8(8): 1274-1284. The sequence of the gRNA expression cassette with 1 target site: SEQ ID NO.1: TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGGaaggaatctttaaacatacgaacagatcacttaaagttcttctgaagcaacttaaagttatcaggcatgcatggatcttggaggaatcagatgtgcagtcagggaccatagcacaagacaggcgtcttctactggtgctaccagcaaatgctggaagccgggaacactgggt acgttggaaaccacgtgtgatgtgaaggagtaagataaactgtaggagaaaagcatttcgtagtgggccatgaagcctttcaggacatgtattgcagtatgggcc ggcccattacgcaattggacgacaacaaaggctagtattagtaccacctcggctatccacatagatcaaagctggtttaaaagagttgtgcagatgatccgtggca GGCGACCGAACAGTTCTTGCAGG gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttcaaGAGCTTGGAGTGGATGGACCCGGTCGAGACCCACGCT; Plasmids were extracted using a kit and digested with Asc I enzyme. The results are as follows: Figure 3 As shown, the vector plasmid was cut out to a length of approximately 564 bp, which is the size of a tandem expression cassette of one U3-gRNA (564 bp), proving that the CRISPR / Cas9 knockout vector has been successfully constructed.

[0018] Example 2. Obtaining and identifying OsMYB71 gene CRISPR-CAS9 transgenic plants The OsMYB71 gene obtained in Example 2 was combined with the recombinant expression vector constructed from pYLCRISPR / Cas9-MT and introduced into a normal Nipponbare rice variety using Agrobacterium EHA105-mediated genetic transformation (Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant Journal 6: 271-282). After the transgenic plants matured, DNA was extracted from leaves, and primer pairs CRISPR-Cas9, 37210-F: CAACGAGATCAAGAACTACTGGAACACG (SEQ ID NO. 2); 37210-R: GGCTTGAGGAGGGAGGTGAGAT (SEQ ID NO. 3), were designed to sequence the target site and identify whether gene mutations occurred near the target site. Figure 4 As shown, positive plants with effective mutations in the OsMYB71 gene were screened, and individual plants were harvested and propagated until homozygous plants were detected in the T2 generation. Two OsMYB71 gene mutant lines were selected from the obtained lines for subsequent experiments and named KO-4 and KO-5, respectively.

[0019] PCR detection was performed using primers Hyg-F / R. Plants with a band size of 229 bp were considered positive, while negative plants could not amplify any bands. Hyg-F: AGAGAAGATGTTGGCGACCTC (SEQ ID NO.4); Hyg-R: GGTATCACTGGCAAACTGTGA (SEQ ID NO.5). Sequencing primers 37210-F / R were then used to amplify the target region sequence of positive plants to identify whether a gene mutation had occurred near the target site. Positive plants with effective gene mutations were screened, and individual plants were harvested and propagated until homozygous plants were detected in the T2 generation.

[0020] CRISPR lines produced mutations near the target site that impaired the normal functioning of the gene. Specifically, the OsMYB71 gene in the KO-4 line had a 3-base deletion at the target site, while the OsMYB71 gene in the KO-5 line had a 1-base insertion at the target site. Figure 5 ).

[0021] Example 3. Obtaining overexpression OsMYB71 Gene carrier Total RNA was extracted from 7-day-old Nipponbare seedlings, and cDNA was obtained by reverse transcription. Then, the OsMYB71 cDNA sequence was amplified by RT-PCR using the cDNA as a template. Figure 1 The specific procedure is as follows: Total RNA was extracted from rice using TRIzol reagent (Invitrogen, Carlsbad, CA). 1 μg of total RNA was then reverse transcribed using a reverse transcription kit manufactured by Hangzhou Xinjing Bio-Reagent Development Co., Ltd., according to the product instructions, to obtain cDNA. Using cDNA as a template, the primer sequences are as follows: Upstream primer 5'tcggtacccgggatccATGGGGCGGGCGCCG3' (SEQ ID NO.6); Downstream primer 5'tgctcaccatacgcgtTTACATGTAGTCGCTCACGTCGAGGAGCT3' (SEQ ID NO.7). In this experiment, an overexpression fusion vector for this gene was constructed based on the CaMV 35S bidirectional driven pCAMBIA1300-35S-EGFP vector. After analysis of the vector sequence and the CDS sequence of the OsMYB71 gene, BamHI and MluI were used to express the gene. Overexpression amplification primers with homologous ends to the restriction enzyme sites were designed to obtain the recombinant expression vector pCAMBIA1300-35S-OsMYB71-EGFP. Figure 6 To determine the success of vector construction, EcoRI and HindIII enzymes were used for digestion. Theoretically, the resulting bands consisted of three parts: 1524 bp, 5630 bp, and 8906 bp. Figure 7 ).

[0022] CDS sequence (SEQ ID NO.8) Protein sequence (SEQ ID NO.9): MGRAPCCEKSGLKKGPWTPEEDEKLIAYIKEHGQGNWRTLPKNAGLSRCGKSCRLRWTNYLRPDIKRGRFSFEEEEAIIQLHSILGNKWSAIAARLPGRTDNEIKNYWNTHIRKRLLRMGIDPVTHAPRLDLLDLTSLLKPAAAAAYYPTQADLDTLRALEPLAGYPDLLRLASAILPAATTTG AAAAAAAEQAQLLLPWLLQAQMAQQQQQVTPPPPPPPPQAAATEQFLQATSTACHQMPGLVHASPTQQLAQQPQDHMAAATCHRRGAVQHPSYDNQLDYVPALMQMASDASNLQQWSSTVSSNNHNVNSGVSTPSSSPAAAGQINSSSTTTTTTYGLNASGDVDDAGLLINMHLSELLDVSDYM.

[0023] Example 4. Obtaining overexpression OsMYB71 Genetically modified rice The recombinant expression vector pCAMBIA1300-35S-OsMYB71-EGFP constructed above was introduced into normal Nipponbare rice cultivar using Agrobacterium EHA105-mediated genetic transformation (Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant Journal 1 6: 271-282). After the transgenic plants grew, DNA was extracted from leaves, and primers Hyg-F / R were designed for PCR. Plants with a band size of 229 bp were positive, while negative plants could not amplify the band. Figure 8 Hyg-F: AGAGAAGATGTTGGCGACCTC; Hyg-R: GGTATCACTGGCAAACTGTGA. Positive plants transformed with the OsMYB71 gene were selected, and individual plants were harvested and propagated until homozygous plants were detected in the T2 generation.

[0024] We also detected the expression levels of OsMYB71 in wild-type and overexpression lines. Seven-day-old seedlings from OsMYB71 overexpression lines and wild-type plants were used. Total RNA was extracted according to the method described in step 1, and cDNA was obtained through reverse transcription. Using this cDNA as a template and the ACTIN1 gene as an internal control, qRT-PCR was performed using a Q-PCR kit (Takara Premix Ex TaqT MII) manufactured by Dalian Baosheng Biotechnology Co., Ltd. Two rice lines overexpressing the OsMYB71 gene were selected and named OE-4 and OE-8. Figure 9 ), for use in subsequent experiments.

[0025] The primer sequences for OsMYB71 qRT-PCR are as follows: upstream primer 5'CGAGAAGAGCGGGCTGAAGA3' (SEQ ID NO.10); downstream primer 5'TTGCCGAGGATGCTGTGGA3' (SEQ ID NO.11); The primer sequences for qRT-PCR of the ACTIN1 gene are: upstream primer 5'TGGTCGTACCACAGGTATTGTGTT3' (SEQ ID NO.12); downstream primer 5'AAGGTCGAGACGAAGGATAGCAT3' (SEQ ID NO.13).

[0026] qRT-PCR system: 0.25 μl 10 μM LP, 0.25 μl 10 μM RP, 0.3 μl cDNA, 4.2 μl double-distilled water, 5 μl SYBR Green reagent. qRT-PCR program: 95℃, 1 min; 95℃, 5 s; 60℃, 30 s, 40 cycles.

[0027] As shown in the figure below, the OsMYB71 gene content in rice lines overexpressing the OsMYB71 gene was higher than that in the wild type, indicating that the OsMYB71 gene overexpression was indeed successful.

[0028] Example 5. Evaluation of alkali tolerance of transgenic lines Rice seedlings at the two-leaf-one-heart stage (WT, overexpression plants, and mutant plants) were subjected to 80 mM NaHCO3 stress for 7 days, and the seedling survival rate, aboveground and underground fresh weight, and dry weight were measured.

[0029] 1. Alkali tolerance of OsMYB71 transgenic material during budding stage The alkali tolerance of transgenic lines during the seedling stage was determined. For example... Figure 10-12As shown, the KO-4 and KO-5 lines at the bud stage showed significantly lower aboveground and belowground performance than the wild type under alkali stress, while the OE-4 and OE-8 lines at the bud stage showed significantly higher aboveground and belowground performance than the wild type under alkali stress. All lengths are in cm, and fresh weight is in g.

[0030] Table 1. Fresh weight of aboveground and underground parts of OsMYB71 transgenic material at bud stage

[0031] 2. Alkali tolerance of OsMYB71 transgenic material during the seedling stage The alkali tolerance of transgenic lines during the seedling stage was determined. For example... Figure 13-20 As shown, the KO-4 and KO-5 lines under alkaline stress had significantly lower plant height, root length, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, chlorophyll content, and survival rate than the wild type, while the OE-4 and OE-8 lines under alkaline stress had significantly higher plant height, root length, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, chlorophyll content, and survival rate than the wild type.

[0032] Table 2. Data on OsMYB71 transgenic materials at the seedling stage

[0033] 3. Physiological index determination of OsMYB71 knockout materials at the seedling stage Physiological indicators used to determine the salt and alkali tolerance of knockout lines during the seedling stage. For example... Figure 10-16 As shown, the activities of superoxide dismutase (SOD) and catalase (CAT), malondialdehyde (MDA) content, proline content, and hydrogen peroxide content of KO-4 and KO-5 seedlings under alkaline stress were measured. DAB and NBT staining was also performed on the KO-4 and KO-5 seedlings under alkaline stress. The results showed that under control and alkaline stress conditions, reactive oxygen species (ROS) indicators were measured in transgenic and wild-type lines. Under control conditions, there was no significant difference between knockout and wild-type materials. However, under alkaline treatment conditions, the DAB and NBT colors of the knockout materials were darker than those of the control, indicating that the knockout materials accumulated more H2O2 and suffered greater oxidative stress losses. The H2O2 content measurement and the detection results of SOD, POD, and CAT enzyme activities also confirmed this conclusion. OsMYB71 Knockout materials reduce ROS accumulation by decreasing SOD, POD, and CAT enzyme activities, thereby reducing rice's alkali tolerance. Meanwhile, under control conditions, there were no significant differences in proline and malondialdehyde (MDA) content between wild-type and transgenic materials. Under alkali treatment, the proline content in knockout materials was lower than that in wild-type materials, while the MDA content was significantly higher. The results indicate that... OsMYB71The alkali resistance of rice is improved by promoting the accumulation of proline and removing malondialdehyde.

[0034] Example 6. Functional evaluation of salt-stressed transgenic materials Rice seedlings at the two-leaf-one-heart stage (WT, overexpression plants, and mutant plants) were subjected to 120 mM NaCl stress for 7 days, and the seedling survival rate, aboveground and underground fresh weight, and dry weight were measured.

[0035] The results are as follows Figure 21 As shown, the KO-4 and KO-5 lines under alkaline stress had significantly lower plant height, root length, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, chlorophyll content, and survival rate than the wild type, while the seedling OE-4 and OE-8 lines under alkaline stress had significantly higher plant height, root length, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, chlorophyll content, and survival rate than the wild type.

Claims

1. The application of OsMYB71 amino acid in regulating the salt and / or alkali tolerance of rice, characterized in that, The amino acid sequence of the OsMYB71 is shown in SEQ ID NO.

9.

2. The application of the OsMYB71 gene in regulating the salt and / or alkali tolerance of rice, characterized in that, The nucleotide sequence of the OsMYB71 gene is shown in SEQ ID NO.

8.

3. The application of a recombinant vector containing the OsMYB71 gene in regulating the rice's tolerance to salt and / or alkali conditions, characterized in that... The nucleotide sequence of the OsMYB71 gene is shown in SEQ ID NO.

8.

4. The application according to claim 3, characterized in that, The starting vector was pCAMBIA1300-35S-EGFP.

5. The application of recombinant microbial cells containing the OsMYB71 gene in regulating the salt and / or alkali tolerance of rice, characterized in that, The nucleotide sequence of the OsMYB71 gene is shown in SEQ ID NO.

8.

6. The application according to claim 5, characterized in that, The starting microbial cells are either prokaryotic or eukaryotic microbial cells.

7. The application according to any one of claims 1-6, characterized in that, The alkali stress condition is 80 mM sodium bicarbonate; the salt stress condition is 120 mM sodium chloride.

8. A breeding method for improving the salt and alkali tolerance of rice, characterized in that, The steps of the method are as follows: Step 1: Amplify the OsMYB71 gene and then ligate it into the pCAMBIA1300-35S-EGFP vector to obtain the recombinant vector; the nucleotide sequence of the OsMYB71 gene is shown in SEQ ID NO.8; Step 2: Transform the recombinant vector obtained in Step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Transfect recombinant Agrobacterium into rice callus and culture to obtain transgenic rice.

9. The breeding method according to claim 8, characterized in that, The primers used to amplify the OsMYB71 gene in step 1 are SEQ ID NO.6 and SEQ ID NO.

7.

10. A method for improving the salt and alkali tolerance of rice, characterized in that, The OsMYB71 gene was overexpressed in rice and cultured under conditions of 80 mM sodium bicarbonate or 120 mM sodium chloride.

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